Characterization of human, mouse, and rat cultures of enteric glial cells and their effect on intestinal epithelial cells.

BACKGROUND: Enteric glial cells (EGC) are major regulators of neuronal and intestinal epithelial cell (IEC) functions. Simple isolation methods of EGC, especially human tissues, remain scarce and limit their study. We present herein a method to isolate EGC and we characterize EGC phenotype and their functional impact on IEC. METHODS: Longitudinal muscle and myenteric plexus preparations of rat, mouse, or human intestine were obtained by microdissection. After mechanical and enzymatic dissociation, individual ganglionic or interganglionic structures were seeded into plates, maintained in culture several weeks and passaged up to 4 times. Purity of cultures was assessed by immunocytochemistry using antibodies against glial fibrillary acidic protein (GFAP), S100ß and Sox10 or smooth muscle actin. Effects of adenosine triphosphate (ATP) on intracellular Ca²âº signaling in EGC were studied. Co-cultures of EGC with IEC line, Caco-2, were performed for 2-6 days to analyze their impact on monolayer resistance, cell proliferation, and cell spreading. KEY RESULTS: More than 80% of DAPI-positive cells were GFAP, S100ß, and Sox10-immunoreactive. EGC expressed these glial markers over 4 consecutive passages, and the majority of them responded to ATP by an increase in intracellular Ca²âº concentration. In addition, rat, mouse, and human EGC increased intestinal barrier resistance, IEC size, and reduced IEC number. CONCLUSIONS & INFERENCES: We have developed a simple method to isolate and culture human, rat, or mouse EGC. EGC exhibit similar functional properties on the intestinal barrier independently of the species. This study sets the basis for exploring glial biology and functions in human health and diseases.

Sacral nerve stimulation enhances epithelial barrier of the rectum: results from a porcine model.

BACKGROUND: The mechanism of action of sacral nerve stimulation (SNS) remains largely elusive. The aims of this study were to develop a clinically relevant animal model for percutaneous SNS and to describe its effect on the epithelial barrier of the rectum. METHODS: Under general anesthesia and after percutaneous electrode placement for S3 nerve root stimulation, six pigs underwent unilateral stimulation and six bilateral stimulation. Animals were stimulated for 3 h using an external pulse generator (1-2.5 V; 14 Hz; 210 µs). Six animals underwent electrode implantation without stimulation and served as controls. Full-thickness rectal biopsies were performed prior to and after stimulation. Paracellular permeability was evaluated by measuring sulfonic acid flux across the rectal mucosa in Ussing chambers. Histological assessment of mucosal thickness, epithelial desquamation, and mucus expression were performed. KEY RESULTS: Percutaneous stimulation resulted in successful anal contractions whose amplitude and uniformity was enhanced following bilateral compared with unilateral stimulation. In controls, paracellular permeability significantly increased during the stimulation period whereas it remained unchanged following unilateral stimulation. In contrast, permeability was significantly reduced by bilateral stimulation. This effect was associated with a concomitant reduction in mucosal thickness and a trend toward increased amount of mucus on surface epithelium compared with controls. CONCLUSIONS & INFERENCES: The development of a porcine model of percutaneous SNS revealed the ability of neuromodulation to reinforce rectal epithelial barrier. Furthermore, our results suggest that SNS could be used for treatment of gastrointestinal pathologies with reduced rectal mucosal barrier functions.